Electrochemical detection has been employed in liquid chromatography and in capillary electrophoresis (CE). It has been demonstrated that electrochemical detection is very sensitive and can measure 10.sup.-16 to 10.sup.-19 moles of sample with typical detection volumes from nL to pL.sup.1,2. Electrochemical methods have also been used to detect DNA,.sup.3-5 single cells,.sup.6,7 and even single molecules..sup.8 The operation of these electrochemical detectors is typically based on the use of three electrodes called the working, counter, and reference electrodes. There are three configurations which have been used to detect CE separations: on-column,.sup.9 where the electrodes of the detector are placed within the capillary; end-column,.sup.10,11 where the electrodes are placed directly at the end of the separation capillary; and off-column,.sup.6,12,13 where the electrodes are electrically isolated from the electrophoresis voltage by a grounded porous glass tube. On-column electrochemical detection of CE separations has been performed by fixing two platinum wires through diametrically opposed holes drilled by a laser in a capillary tube. This structure is very difficult to manufacture and align, and the placement of the detection electrodes within the high voltage region of the separation column is problematic. In this format, one is trying to detect small currents or voltages while applying many kV to the separation column. The mechanical instability and poor definition of the electrode alignment can lead to significant electrical pickup or fluctuation in the background, making the desired signal very difficult to detect. The presence of high voltage gradients and significant electrophoretic currents in the column near the electrodes can induce stray signals. The end-column and off-column detection formats are important because they minimize the influence of the electrophoresis voltage. In the end-column format, one wants to place the detection electrodes as close to the end of the electrophoresis channel as possible so the detection is performed as close to ground potential as possible. This is very difficult to do with conventional manufacturing techniques. The electrodes must be placed with micron precision at the end of the capillary. Any error in the placement will cause loss of analyte signal if the electrodes are too far from the opening or high voltage pick up if the electrodes are placed within the separation column. Furthermore, fluctuations in electrode placement or electrode--electrode gap can cause severe fluctuations in the background signal producing noise. Typically, one must use micromanipulators and a microscope to assemble the detector. Furthermore, the engineering of the electrical isolation by connection of the separation and detection capillary tubes with a grounded porous glass tube in the off-column format is rather difficult to assemble and operate, and the junction can be mechanically unstable and poorly defined. In one case, although Slater and Watt (17) photolithographically fabricated electrodes on a substrate, because they did not make a fully integrated separation and detection device, they were forced to use said undesirable junctions to couple their detector to a conventional cylindrical capillary.
There is a need for a microfabricated capillary electrophoresis chip with integral thin film electrochemical detector and electrophoresis leads which can be easily connected to associated electrical electrophoresis and detector apparatus.